April Davis

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by Expanding the Cancer Stem Cell Population

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy of Adjuvant Trastuzumab

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

Notch Reporter Activity in Breast Cancer Cell Lines Identifies a Subset of Cells with Stem Cell Activity

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch−) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch− cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.

SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer.

Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)–deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display “cancer stem cell” properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Experimental Design: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1–wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1–wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22. ©2016 AACR.

A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab-Resistant Tumors

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance.

Abstract 2311: Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways

Cancer stem cells (CSCs) drive tumor growth as well as mediating metastasis and treatment resistance. In breast cancer (BC), CSCs exist in alternative mesenchymal (EMT) and epithelial (MET)-like states characterized by the expression of CD44+CD24- and aldehyde dehydrogenase (ALDH) respectively. BCSCs display phenotypic plasticity allowing them to transition between EMT and MET states in a process regulated by the tumor microenvironment. The plasticity of BCSCs suggests that it may be necessary to simultaneously target alternative BCSC states to achieve maximal eradication of these cell populations. In order to develop strategies to target these BCSC states, we measured the cellular bioenergetics of EMT and MET BCSCs compared to non-stem bulk tumor cells as well as the sensitivity of distinct BCSC states to inhibitors of glucose and hydroperoxide metabolism. Using a Seahorse XCF instrument, we found that both MET and EMT BCSCs display higher glycolytic potential than bulk tumor cells. Increased glycolysis correlated with elevated expression of hexokinase 2, a rate limiting glycolytic enzyme in cancer. Interestingly, the glycolytic inhibitor, 2-deoxyglucose (2DG), specifically inhibited EMT BCSCs in a dose dependent fashion; however, MET BCSCs were completely refractory to this treatment. Proteomic and RNA-seq analyses revealed that two important arms of hydroperoxide metabolism, the thioredoxin- and glutathione-mediated antioxidant pathways, were robustly up-regulated in MET BCSCs, suggesting that these cells are rendered resistant to glycolysis inhibition via an enhanced anti-oxidant defense. Pharmacologic inhibition of this antioxidant defense by Auranofin (an inhibitor of thioredoxin reductase) is sufficient to deplete MET BCSCs in SUM149 BC cells which were rescued by NAC, a ROS scavenger, or catalase, a specific enzyme capable of degrading intracellular H2O2. Finally, administration of 2DG together with Auranofin and BSO (an inhibitor of glutathione metabolism) synergistically suppressed tumor growth in a patient derived xenograft (PDX) model by suppressing both EMT and MET BCSCs. This study suggests that utilizing metabolic inhibition to simultaneously target EMT and MET BCSCs is a viable therapeutic strategy with important clinical implications. Citation Format: Ming Luo, April Davis, Sean McDermott, Evelyn Jiagge, Michael Brooks, Elizabeth Gheordunescu, Tahra Luther, Shawn G. Clouthier, Sarah Conley, Douglas R. Spitz, Max S. Wicha. Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2311. doi:10.1158/1538-7445.AM2015-2311

Abstract 4226: Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer

Background: Triple negative breast cancers (TNBCs) have a poor prognosis and are a therapeutic challenge due to resistance to multiple chemotherapy drugs. Growing evidence suggests that cancer stem cells (CSCs) may be promising therapeutic targets for treating TNBCs. Poly ADP ribose polymerase (PARP) inhibitors have shown striking activity in preclinical models of BRCA-deficient breast carcinomas. However they are less efficacious in tumors without germline BRCA mutations, which account for more than 80% of all TNBCs. Our data demonstrates that treatment of TNBC with PARP inhibitor increases CSCs, likely due to an elevated homologous recombination (HR) repair mechanism. Several small studies have suggested that histone deacetylase (HDAC) inhibitors, which decrease HR repair proteins, may sensitize non-BRCA-mutated TNBC to PARP inhibitors. Currently, little is known whether this synergistic effect can reverse the increase in CSCs caused by PARP inhibition. In this study, our aim was to investigate if HDAC inhibitors could sensitize CSCs to PARP inhibitors in TNBCs. Results: Using four TNBC cell lines, we confirmed the synthetic lethality of HDAC and PARP inhibitors (Vorinostat and Olaparib respectively) in vitro. CompuSyn analysis revealed Combination Indices of 0.66, 0.44, 0.47 and 0.16 in SUM159, MDAMB231, SUM149 and HCC1937 cells respectively at ED50. An ALDEFLUOR assay revealed ∼1.9 fold increase in the CSC population after PARP inhibition in BRCA-mutant SUM149 and HCC1937 cells, while the absolute CSC number, (total cell number times percentage of ALDEFLUOR-positive cells) remained unchanged. Whereas in BRCA-wild type TNBC cell lines SUM159 and MDAMB231, there was no change in CSCs. Addition of the HDAC inhibitor decreased absolute CSC number by 85%, 78% and 40% in SUM149, SUM159 and HCC1937 cells respectively after 7 days of PARP inhibition. Although the mechanism is not fully understood at present, we demonstrated that Rad51, a key player in HR repair, mediates the sensitivity of TNBCs to PARP inhibition, and HDAC inhibitor prevents the formation of Rad51 foci at DNA damage site. This may indicate that HDAC inhibition sensitizes the CSCs of TNBCs to PARP inhibition via suppressing the HR pathway. Conclusion: Our data suggests that PARP inhibition only targets the bulk cancer cells in BRCA-mutant TNBC cells while sparing CSCs and has no effect on BRCA WT cells. HDAC inhibition sensitized CSCs of TNBC to PARP inhibition regardless of BRCA status, possibly via interfering with Rad51 function. The inhibitors we used in the present study are either FDA-approved or at Phase III clinical trials, and the combination is predicted to have low toxicity. Thus, our findings are promising and could be rapidly translated into clinical use. Citation Format: Yajing Liu, Rachel Martin-Trevino, Li Shang, April Davis, Max Wicha, Suling Liu, Monika Burness. Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2015-4226

Abstract 4119: The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism

EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism. Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 15% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This causes a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggests that the C>T mutation is an acquired event secondary to cytosine deamination by Activation Induced Cytosine Deamination enzyme (AICDA). Results: Single cell clones of lung adenocarcinoma cell line, PC9, were treated with EGFR TKI. At baseline, these clones have EGFR exon 19 deletion but no evidence of T790M mutation by digital droplet PCR (ddPCR). However, after treatment with a serial increasing dose of EGFR TKI, T790M mutation was detected by ddPCR. Assessing whether cytosine deamination enzymes were altered by this treatment, a significant increase in AICDA expression was seen. Furthermore, recombinant AICDA protein could deaminate cytosine at position 2369 in vitro. In addition, using mass spectrometry and methylation specific primers, we determined that cytosine at position 2369 is in fact methylated. This further supports our hypothesis since 5-methyl cytosine is deaminated into thymine directly. Since in germinal center B-lymphocytes, AICDA is activated through a non-canonical NFkB mechanism, we assessed NFkB pathway in PC9 cell line. RelB and p52 expression were significantly increased after TKI treatment. In addition direct interaction between RelB and AICDA promoter was confirmed by ChIP Assay. These findings were also seen in a mouse PC9 xenograft model. Daily oral gavage of EGFR TKI caused significant increase in the expression of RelB as well as AICDA. Adding NFkB inhibitor twice weekly inhibited the expression of RelB and AICDA. Finally, knocking down AICDA by shRNA prevented the development of T790M mutation in PC9 cell lines after TKI exposure. Conclusion: Our data suggest that the T790M mutation could be actively acquired after TKI treatment through a cytosine deamination process by AICDA. This would have significant implications for treatment with targeted therapy. In fact, Imatinib resistance in CML and GIST tumors have a similar C>T single nucleotide transition mutation. Citation Format: Khaled Hassan, Najwa El Kadi, April Davis, Gregory Kalemkerian, Luo Wang, Hasan Korkaya. The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2017-4119

Abstract 3866: Inactivation of p53/PTEN confers a specific epigenetic profile regulated by IL6-SOCS3 signaling

Recent studies by us and others suggested that activation of inflammatory feedback loop play critical role in metastasis and therapeutic resistance by regulating EMT/CSC phenotype. Of these cytokines, IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Stat3/NF-kB pathway which is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). Consistent with these reports, we demonstrated in our preliminary data that SOCS3 is abundantly expressed in non-malignant MCF10A cells as well as in luminal and HER2+ tumors where it negatively regulates the IL6/Stat3/NF-kB signaling. In contrast, SOCS3 protein was undetectable in basal cluadin-low breast cancer cell lines and primary tumors which express substantially higher levels of IL6. We developed transformed MCF10A model by simultaneous knockdown of p53 and PTEN. In this model single p53 and PTEN deletions result in hyperplasia or DCIS-like lesions. Interestingly, SOCS3 protein expression was undetectable in MCF10A-p53-PTEN- cells compared to parental MCF10A, single p53 (MCF10A-p53-) or PTEN (MCF10A-PTEN-) deleted cells. We demonstrated that IL6 mediated Stat3/NF-kB signaling is negatively regulated by SOCS3 that strongly inhibited the IL6/Stat3/NF-kB pathway when overexpressed in MCF10A-p53-PTEN- cells. Moreover, enforced SOCS3 expression in these cells reduced the tumor growth and inhibited metastasis. Furthermore, loss of SOCS3 expression is strongly associated with increased risk of recurrent disease in breast cancer patients. We hypothesized that loss of SOCS3 in TNBCs results in activation of IL6 feedback loop which drives aggressive metastatic phenotype. Interestingly Stat3/NF-kB signaling drives expression of SOCS3 in response to inflammation and serves as negative feedback loop, although molecular mechanism of SOCS3 expression in cancer has not been investigated. IL6 signaling has been previously reported to be involved in epigenetic regulation. We therefore correlated the gene expression and methylation profile after enforced SOCS3 expression. Our analysis revealed that expression levels of a significant number of genes involved in transcription, migration and metabolism were reverted to those of the parental non transformed cells and their transcription was regulated by methylation. These results suggest that interplay between genetic and epigenetic changes during malignant transformation of mammary epithelial cells may activate multiple signaling cascades and reconstitution of SOCS3 may have therapeutic utility in TNBC. Citation Format: Maria Ouzounova, Gwangil Kim, April Davis, Ahmed A. Quraishi, Nader Tawakkol, Shalini Kota, Max S. Wicha, Hasan Korkaya. Inactivation of p53/PTEN confers a specific epigenetic profile regulated by IL6-SOCS3 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2014-3866